No Difference in Intestinal Strontium Absorption After an Oral or an Intravenous 1,25(OH)₂D₃ bolus in Normal Subjects

INTRODUCTION

The clinical use of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in chronic renal failure is often limited by hypercalcemia. Calcitriol pulse therapy is considered an important part of the standard treatment of secondary renal hyperparathyroidism, since it has been shown to suppress parathyroid cell proliferation, thus preventing parathyroid gland hyperplasia,¹ and to reduce parathyroid hormone (PTH) gene transcription.² It has been postulated that intravenous (iv) therapy with 1,25(OH)₂D₃ is more effective than oral treatment in decreasing PTH plasma levels.^{3, 4} At the same time, iv 1,25(OH)₂D₃ is believed to decrease the frequency of hypercalcemia by avoiding direct contact with the enterocytes during the intestinal calcium absorption process.³ However, no direct study addressing the question as to whether iv and oral 1,25(OH)₂D₃ have different effects on intestinal calcium absorption has been published. To evaluate intestinal calcium absorption in response to a single oral or iv dose of 1,25(OH)₂D₃, we measured it in healthy volunteers, using stable strontium (Sr) as a surrogate tracer for calcium.⁵ A cross-over design was used, with adequate wash-out periods between tests.

MATERIALS AND METHODS

Study protocol

Each subject was submitted to five separate Sr absorption tests with intervals of at least 21 days (wash-out periods), during which the serum Sr concentration was allowed to return to baseline values. The five tests are identified by the letters A–E: test A, Sr absorption without 1,25(OH)₂D₃; test B, Sr absorption immediately after administration of oral 1,25(OH)₂D₃; test C, Sr absorption immediately after administration of iv 1,25(OH)₂D₃; test D, Sr absorption, 24 h after oral 1,25(OH)₂D₃; and test E, Sr absorption, 24 h after iv 1,25(OH)₂D₃.

A cross-over design was chosen for the study. Test A was always performed as the first test, whereas tests B–E, were performed in a random sequence.

All subjects had been instructed to maintain a self-selected diet containing a normal calcium intake (assessed through an interview with the dietician) throughout the week before and during the study period, but to avoid toothpaste in the 12 h preceding the test, since toothpaste often contains Sr.

For each test, the subject was admitted to the hospital in the early morning after an overnight (12 h) fast. Weight and height were recorded, and an in-dwelling catheter was placed in a forearm vein. Blood samples were drawn for baseline laboratory evaluation: serum calcium, phosphorus, creatinine, ALP, albumin, bicarbonate, BUN, 1,25(OH)₂D₃, and plasma iPTH (Table 1).

Table Table 1. Study Protocol

At this point, in test A (Sr absorption without 1,25(OH)₂D₃, the Sr absorption test, as described below, was started immediately. In tests B and C, 1,25(OH)₂D₃ was administered per os or iv respectively (see below), then the Sr absorption test was started immedately. In tests D and E, 1,25(OH)₂D₃ was administered per os or iv, respectively, then, after 24 h, the Sr absorption test was performed. In all tests (A–E), serum calcium, phosphorus, 1,25(OH)₂D₃, and plasma iPTH concentrations were measured at 1, 2, 3, and 4 h. In tests D and E, serum calcium, phosphorus, 1,25(OH)₂D₃, and plasma iPTH concentrations were also measured 6, 12, 24, 48, and 72 h after 1,25(OH)₂D₃ administration. In tests C and E, blood was also drawn at 5 minutes after iv 1,25(OH)₂D₃ for serum 1,25(OH)₂D₃ determination. The protocol is summarized in Table 1.

For the serum determinations, blood in tubes was allowed to clot for 30 minutes at room temperature before centrifugation. All serum and plasma samples were frozen immediately after centrifugation and stored at −20°C until analysis.

The study was approved by the Institutional Review Board of the Department of Pediatrics, University of Milan and the Ethical Committee of the University of Heidelberg. Written informed consent was obtained from each volunteer.

Statistical analysis

Sr concentration is expressed as the fraction of the adsorbed dose (FAD%) contained in the extracelluar fluids (assumed equal to 15% of ideal body weight), according to the formula⁵:

For all variables, the Shapiro–Wilk's test for normality was used⁶ in order to choose the appropriate statistical analysis. All the studied variables were found to be normally distributed within time and groups. The baseline descriptive variables are given as mean ± SD. The values of the variables measured over time (1,25(OH)₂D₃, Sr, and iPTH) are presented as least squares means ± SE.⁸

Analysis of variance was used to evaluate baseline values for Sr, calcium, phosphorus, iPTH, 1,25(OH)₂D₃, maximum Sr concentration (C_max), and the area under the Sr FAD%–time curve over the 240 minutes (SrAUC_0–240). The AUCs were calculated using the trapezoidal rule (Bezout integral); the computer-calculated areas were rounded.

The general linear model for unbalanced data (Proc. Glm SAS/PC version 6.04; SAS Institute, Cary, NC, U.S.A.) with a split-plot design was used to compare the effects of different administration routes of 1,25(OH)₂D₃ at different times. The means calculated within this model (population marginal means) are obtained by the least squares method and are the expected values of class or subclass means that would be expected for a balanced design. The standard error in this analysis is referred to the mean value for the general linear model. The linear and quadratic trends between and within groups were evaluated using orthogonal polynomial fitting especially constructed for data unequally spaced in time. According to Scheffé's test, multiple within-group comparisons were performed when the linear or quadratic trends were significant.^{7, 9} A p value ≤ 0.05 was considered statistically significant. All of the statistical tests were two sided.

RESULTS

All the subjects had normal serum calcium, phosphorus, 1,25(OH)₂D₃, and plasma iPTH levels at baseline. The mean basline level of 1,25(OH)₂D₃ was 33.8 ± 6.4 pg/ml (range 18–50 pg/ml), and that of iPTH was 28.1 ± 12 pg/ml (range 13.7–58.0).

The 1,25(OH)₂D₃ serum concentration versus time after oral and iv 1,25(OH)₂D₃ administration is shown in Fig. 1. As expected, the kinetics of 1,25(OH)₂D₃ were substantially different between the two administration modalities. The peak 1,25(OH)₂D₃ concentration (oral: 101 ± 15 pg/ml; iv: 297 ± 24 pg/ml; p < 0.001) but also the area under the 1,25(OH)₂D₃ concentration–time curve (oral: 3180 ± 291 pg/ml · h; iv: 4260 ± 429 pg/ml · h; p < 0.01) were significantly higher after iv administration. Six hours after the administration of the 1,25(OH)₂D₃, the serum concentration of 1,25(OH)₂D₃ was no longer significantly different in the two groups.

Figure 2 shows Sr fractional absorption over time in the five tests. When oral or iv 1,25(OH)₂D₃ was administered at the start of the Sr absorption test (tests B and C), Sr fractional absorption was not changed with respect to test A, where no 1,25(OH)₂D₃ was given. On the contrary, Sr absorption measured 24 h after 1,25(OH)₂D₃ administration was significantly increased with both the oral (test D) and the iv (test E) route. Similar findings were observed when the results were analyzed as the area under the Sr fractional absorption–time curve and in terms of peak Sr concentration (Table 2). Again, there was no difference between the oral or iv 1,25(OH)₂D₃ administration routes.

Table Table 2. Strontium Intestinal Absorption Tests in Normal Subjects Under Different Conditions with Regard to the Route and Timing of Administration of 1,25(OH)₂D₃

Neither serum calcium and phosphorus levels nor plasma iPTH levels showed any significant changes in relation to the 1,25(OH)₂D₃ administration route. In particular, the mean of the maximum concentrations of serum calcium and phosphorus levels after oral or iv 1,25(OH)₂D₃ were, respectively, 9.87 ± 0.37 mg/dl and 9.78 ± 0.31 mg/dl (calcium) and 4.47 ± 0.10 mg/dl and 4.24 ± 0.22 mg/dl (phosphorus); the mean iPTH minimum concentrations were 15.7 ± 4.8 pg/ml and 16.7 ± 3.1 pg/ml.

DISCUSSION

The present study demonstrates that, in normal human subjects, a pharmacological dose of 1,25(OH)₂D₃ increases Sr absorption (used as a surrogate for measuring calcium absorption) by 35% as measured 24 h after 1,25(OH)₂D₃ administration, whereas no early increase in Sr absorption could be detected immediately after use of the drug. The study also demonstrates for the first time that the increase in intestinal calcium absorption is not influenced by the route of administration of 1,25(OH)₂D₃.

The use of stable Sr to investigate intestinal calcium absorption deserves comment. Calcium and Sr actually compete for intestinal absorption,¹⁰ bone distribution,¹¹ and renal excretion,¹² and their metabolism is controlled by the same hormones.¹³ According to the periodic table, Sr is the most similar element to calcium.¹⁴ Moreover, Sr shares a number of other common characteristics with calcium: it mimics calcium in many biochemical reactions, it can substitute for calcium in transport systems, and it shares with calcium the process for uptake in the mitochondria. The high Sr affinity for calcium-binding proteins, although less than that of calcium in absolute value,^15-17 theoretically makes it a reliable means of measuring intestional calcium absorption,^18-22 especially in a comparative analysis. Reid et al.⁵ who first developed the test as presently used, demonstrated a very close correlation (r = 0.93) between Sr and calcium absorption as comeasured by⁴⁵Ca: the fractional absorption of Sr was lower than that of⁴⁵Ca, but its time course curve closely resembled that of radioactive calcium. The same conclusion was reached by Milson et al., who performed the test under different clinical conditions, characterized by abnormalities in calcium absorption.²³ The stable Sr test is, therefore, currently considered an easy, safe, and reliable method for estimating the intestinal absorption of calcium.^{24, 25} Especially in the present research setting, in which repeated measurements were made in young subjects—including females of reproductive age—over a short time span, the absence of radioactivity in stable Sr makes it preferable to⁴⁵Ca for estimating calcium absorption.

1,25(OH)₂D₃ is reported to influence the intestinal calcium absorption in vertebrates with at least two separate actions: a rapid (nongenomic) action on the cellular membranes and transport systems and a delayed action which involves gene activation and protein synthesis.^{26, 27} There appear to be several different mechanisms ensuring calcium entry across the brush-border membrane into the intestinal cell, as well as its translocation through the cytoplasm and its release across the basal lateral membrane into the circulation.^28-31 In vitamin D–deficient animals, a biphasic response has been observed after a single iv dose of 1,25(OH)₂D₃: the first phase begins within 2 h and peaks by 6 h; the second phase begins after 12 h and peaks at 24 h ³² In vitamin D–replete animals, the “rapid” (nongenomic) action of 1,25(OH)₂D₃, not fully explained yet, is called transcaltachia and develops within a few minutes.³³ It seems to involve the opening of membrane calcium channels and calcium transport through the cell in lysosome-like vesicles, with subsequent release in the circulation.³⁴

The main action of 1,25(OH)₂D₃ on intestinal calcium absorption, however, requires several hours to develop, depending on the activation of nuclear vitamin D receptors (VDRs), gene transcription, and protein synthesis; the membrane calcium pumps are potentiated, and the intracellular calcium is taken up by newly synthesized proteins (calbindins) and actively transported toward the capillaries across the epithelial cells.^{35, 36}

In our subjects, we measured intestinal Sr absorption with separate tests, after either oral or iv 1,25(OH)₂D₃ in order to study both the early and the delayed actions of the drug. However, during the first 4 h after either oral or iv administration of calcitriol, no “early” induction of intestinal Sr absorption was detected. It thus seems possible that transcaltachia is not very relevant in ostensibly healthy young adults on self-selected diets.

On the contrary, substantial stimulation of Sr absorption (> 35%) was observed 24 h after 1,25(OH)₂D₃ administration. The increase was in the same range as that observed in other studies in which, 8–16 h after calcitriol, calcium transport was increased by ∼25% and the net calcium absorption by 33%.^{37, 38} The observation of a significant increase in the intestinal absorption of Sr 24 h after 1,25(OH)₂D₃ administration is consistent with the delayed mechanism of action of calcitriol, i.e., the increase of calcium transport as a result of genomic effects (synthesis of calbindins and other proteins).³⁸

The most interesting finding of our study was the fact that the route of 1,25(OH)₂D₃ administration did not affect intestinal Sr absorption. In 1984, Slatopolsky et al.³ favored the use of iv 1,25(OH)₂D₃ in the treatment of renal patients with secondary hyperparthyroidism, since the use of oral 1,25(OH)₂D₃ was limited by hypercalcemia and hyperphosphatemia. It was postulated that intermittent iv therapy with 1,25(OH)₂D₃ was superior to oral administration as a result of a number of mechanisms: higher peak concentration leading to greater occupancy of the parathyroid VDR, increased bioavailability through circumvention of the “first-pass” hepatic degradation of 1,25(OH)₂D₃; and decreased frequency of hypercalcemia and hyperphosphatemia by the avoidance of direct contact between 1,25(OH)₂D₃ and intestinal epithelial cells.^{3, 4}

No previous study specifically addressed this last point in human subjects; as a result of the present study, Slatopolsky's hypothesis of a significant difference in the stimulation of intestinal calcium absorption between the two routes of administration of 1,25(OH)₂D₃ is not supported, at least in healthy subjects. Moreover, while both routes of administration of 1,25(OH)₂D₃ induced a decrease in iPTH levels, no different effects on iPTH were observed between them in our normal subjects.

In conclusion, our study does not support the view that intestinal calcium absorption is influenced by the route of 1,25(OH)₂D₃ administration. However, the study was conducted in normal subjects, and whether this also holds true for patients with chronic renal failure and secondary hyperparathyroidism remains to be demonstrated.